Diseases that jeopardize or weaken skeletal function constitute the most prevalent chronic impairments in the U.S with associated annual direct and indirect costs of $849 billion. As the baby boomer generation ages, this figure is expected to escalate in the next 10-20 years. Therefore, new strategies that stimulate skeletal repair and regeneration are badly needed. The current literature indicates that the Wnt pathway is among the most attractive candidates for enhancing skeletal repair and regeneration. Disruptions in the Wnt pathway are associated with multiple human musculoskeletal diseases and Wnt signaling is part ofthe body's natural response to skeletal damage. Thus, the objective of this proposal is to examine the basic mechanism(s) by which Wnt signaling effect bone regeneration and to use this information to formulate therapeutic strategies for bone repair. In Aim 1, we will examine how the loss of the Wnt inhibitor, Axin2, affects Wnt signaling in a cell. We hypothesize that the loss of Axin2, will result in an increased in the duration and magnitude of Wnt response. We will examine the Wnt response in primary osteoprogenitor cells isolated from Axin2 mutant mice that has been transfected with Wnt reporter lentivirus where luciferase activity is the readout of Wnt signaling. In Aim 2, we will examine the effect of Axin2 mutation on the osteogenic potential of bone marrow cells both in vitro and in vivo. We hypothesize that loss of Axin2 increases the osteogenic potential of bone marrow stromal cells (BMSC). In our first set of experiments we will evaluate osteoblast differentiation of wild-type and Axin2 mutant BMSCs with or without purified Wnt3a protein. In addition, we will test the osteogenic potential of bone marrow cells from Axin2 mice in vivo by transplanting them into critical size defects in calvaria. We will examine cell proliferation, osteogenic differentiation, and matrix deposition at the injury site. In Aim 3, we will use Wnt protein itself as a therapeutic agent and we will determine its affect on bone healing and regeneration. Similar to Aim 2, Wnt3a or PBS liposomes treated whole bone marrow will be grafted into critical size defects in the calvaria. We will use in situ hybridization, u-CT, histology and immunohistochemistry to characterize the effects of the treatment on cell proliferation, osteoblast differentiation, and matrix deposition in the healing tissue. In summary, these proposed experiments will clarify the function of Axin2 in Wnt signaling, and will directly test the feasibility of using Wnt to stimulating bone regeneration. We envision that this information will be of considerable value in therapeutic approaches to treating musculoskeletal conditions.